Sensory rhodopsin I (SR-I) is a phototaxis receptor in the archaeon Halobacterium salinarium. The receptor protein is similar in structure to visual pigments, consisting of a single polypeptide which folds into 7- membrane-spanning alpha-helical segments forming an internal pocket where the chromophore retinal is bound. A second integral membrane protein, Htrl, which exhibits sequence homology with eubacterial chemotaxis transducers, is essential for SR-I signaling, and there is compelling but indirect evidence for the presence of SR-I and Htrl in a molecular complex. Our goal is to understand the mechanism of SR-l/Htrl coupling during phototaxis signaling. Experiments are designed to analyze the physical association and structural features of SR-I and Htrl in native membranes by spectrophotometric and protein quantitation to assess SR-I to Htrl stoichiometry; sulfhydryl engineering to probe transmembrane topology of both proteins and their oligomeric states in the dark and after photoactivation; deletion analysis and localized random mutagenesis combined with selection for phototaxis-deficient mutants and intergenic suppressors to define the interacting surfaces of the proteins. Knowledge gained will be applied to in vitro experiments, including interaction of Htrl and active fragments with purified SR-I. High yield purification methods are being developed for these experiments as well as to support crystallization efforts. To provide a comparison to SR-I and Htrl interaction, identification and cloning of related sensory receptors and transducers in the cell will be pursued. Photoconversion of SR-I to its signaling state is accompanied by proton transfer reactions initiated in its photoactive center. In the absence of Htrl, SR-I photoreactions result in light-driven electrogenic proton ejection from the cell. This proton pumping is suppressed by interaction with Htrl which blocks proton release. These results suggest that light- induced proton transfer to Htrl or to a site on the receptor coupled to Htrl is an important step for signal transduction. This hypothesis will be tested by site-specific mutagenesis of protonatable residues in SR-I likely to participate in proton transfers within the molecule and possibly to Htrl. Mutants will be characterized for phototaxis signaling in vivo. The 7-transmembrane helix motif is characteristic of a large family of membrane receptor proteins which sense light, hormones neurotransmitters, and chemotaxis stimuli in humans and analogous photo-and chemo-stimuli in microorganisms. A fundamental question in 7-helix sensors is how the activation of the receptor by photon absorption or ligand binding is communicated to its transducer. Because Htrl modulates SR-I photoreactions we have assays available for the interaction based on kinetic flash spectroscopy. This and the availability of genetic methods provides an exceptional opportunity to understand the chemistry of signal relay from a 7-helix receptor to its transducer. Principles elucidated are likely to be relevant to visual pigments and other 7-helix receptors. Additionally, the SR-I transducer is eubacterial in origin and this novel receptor/transducer combination may reveal a new mechanism of signal transduction.